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Free Radic Biol Med. 2016 Feb;91:178-87. doi: 10.1016/j.freeradbiomed.2015.09.011. Epub 2015 Oct 9.

NADPH-cytochrome P450 reductase-mediated denitration reaction of 2,4,6-trinitrotoluene to yield nitrite in mammals.

Author information

1
Environmental Biology Laboratory, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan; Graduate School of Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
2
Graduate School of Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
3
Doctoral Program in Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
4
Application and Research Center, JEOL Ltd., 3-1-2 Musashino, Akishima, Tokyo 196-8558, Japan.
5
Graduate School of Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan; Doctoral Program in Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
6
Environmental Biology Laboratory, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan; Graduate School of Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan; Doctoral Program in Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Electronic address: yk-em-tu@md.tsukuba.ac.jp.

Abstract

While the biodegradation of 2,4,6-trinitrotoluene (TNT) via the release of nitrite is well established, mechanistic details of the reaction in mammals are unknown. To address this issue, we attempted to identify the enzyme from rat liver responsible for the production of nitrite from TNT. A NADPH-cytochrome P450 reductase (P450R) was isolated and identified from rat liver microsomes as the enzyme responsible for not only the release of nitrite from TNT but also formation of superoxide and 4-hydroxyamino-2,6-dinitrotoluene (4-HADNT) under aerobic conditions. In this context, reactive oxygen species generated during P450R-catalyzed TNT reduction were found to be, at least in part, a mediator for the production of 4-HADNT from TNT via formation of 4-nitroso-2,6-dinitrotoluene. P450R did not catalyze the formation of the hydride-Meisenheimer complex (H(-)-TNT) that is thought to be an intermediate for nitrite release from TNT. Furthermore, in a time-course experiment, 4-HADNT formation reached a plateau level and then declined during the reaction between TNT and P450R with NADPH, while the release of nitrite was subjected to a lag period. Notably, the produced 4-HADNT can react with the parent compound TNT to produce nitrite and dimerized products via formation of a Janovsky complex. Our results demonstrate for the first time that P450R-mediated release of nitrite from TNT results from the process of chemical interaction of TNT and its 4-electron reduction metabolite 4-HADNT.

KEYWORDS:

2,4,6-trinitrotoluene; Denitration; Enzyme purification; Metabolism; NADPH-cytochrome P450 reductase; Reactive oxygen species; Xenobiotic

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